CN110499301A - A kind of meso-diaminopimelate dehydrogenase mutant that catalytic efficiency improves - Google Patents
A kind of meso-diaminopimelate dehydrogenase mutant that catalytic efficiency improves Download PDFInfo
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- CN110499301A CN110499301A CN201910807954.6A CN201910807954A CN110499301A CN 110499301 A CN110499301 A CN 110499301A CN 201910807954 A CN201910807954 A CN 201910807954A CN 110499301 A CN110499301 A CN 110499301A
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- C12Y104/01016—Diaminopimelate dehydrogenase (1.4.1.16)
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Abstract
The invention discloses meso-diaminopimelate dehydrogenase mutant that a kind of catalytic efficiency improves, and belong to gene engineering technology field.The present invention carries out single-point/double-site mutant by the meso to SEQ ID NO.2-diaminopimelate dehydrogenase, and successful expression and mutant enzyme D94A is purified into Escherichia coli, W123K, D94A/W123K, W148K, pass through enzyme activity determination, it was found that the three kinds of mesos-diaminopimelate dehydrogenase mutant has relative to the higher catalytic activity of wild type, such as mutant enzyme DAPDH-D94A, DAPDH-W123K, the efficiency of DAPDH-D94A/W123K catalysis phenylpyruvic acid has been respectively increased 3.5, 1.5, 1.3 again, successfully improve the enzyme activity of key enzyme.
Description
Technical field
The present invention relates to meso-diaminopimelate dehydrogenase mutant that a kind of catalytic efficiency improves, and belong to gene
Field of engineering technology.
Background technique
D-phenylalanine food, industry, agricultural, in terms of have very big application value, such as controlling
Treat strontium ranelate dry suspension, the nateglinide tablet for treating diabetes etc. of diabetes.Meso-diaminopimelic acid is de-
Hydrogen enzyme (DAPDH, EC 1.4.1.16) have been widely used for D type aromatic amino acid such as D-phenylalanine preparation (Akita H,
et al.Highly stable meso-diaminopimelate dehydrogenase from an Ureibacillus
thermosphaericusstrain A1 isolated from a Japanese compost:purification,
characterization and sequencing.AMB Express,2011,1:43).And existing part research internally disappears
Rotation-diaminopimelate dehydrogenase has carried out its Fixedpoint mutation modified property, transformation is all concentrated to be located near substrate catalysis region,
Enzyme surface or specific amino acid residue, such as Parmeggiani F selected 150 glutamine, 154 aspartic acids,
169 tyrosine, 195 arginine and 244 hyte propylhomoserins are mutated (Parmeggiani F, et al.Singlel
biocatalyst Synthesis of Enantiopure D-Arylalanines Exploiting an Engineered
D-Amino Acid Dehydrogenase.Advanced Synthesis&Catalysis,2016,358(20):3298-
3306.) successfully it is changed by high substrate specificity, while also being worked to aromatic series ketone acid such as phenylpyruvic acid.But mesh
Before do not have studies have reported that being transformed meso-diaminopimelate dehydrogenase substrate channels to improve enzyme activity.
Summary of the invention
The first purpose of the invention is to provide a kind of meso-diaminopimelate dehydrogenase mutant, the mutation
Body is the 94th aspartic acid to be replaced with alanine, or/and the 123rd tryptophan is dashed forward on the basis of SEQ ID NO.2
Become lysine.
In one embodiment of the invention, the mutant is on the basis of SEQ ID NO.2, by the 94th day
Aspartic acid replaces with alanine, obtains mutant Asp94Ala, i.e. D94A, contains amino acid sequence shown in SEQ ID NO.4.
In one embodiment of the invention, the mutant is on the basis of SEQ ID NO.2, by the 123rd
Tryptophan is mutated into lysine, obtains mutant Trp123Lys, i.e. W123K, contains amino acid sequence shown in SEQ ID NO.6
Column.
In one embodiment of the invention, the mutant is on the basis of SEQ ID NO.2, by the 94th day
Aspartic acid replaces with alanine, and the 123rd tryptophan is mutated into lysine, mutant D94A/W123K is obtained, contains SEQ
Amino acid sequence shown in ID NO.8.
A second object of the present invention is to provide the genes for encoding the mutant.
In one embodiment of the invention, mutant D94A gene as shown in SEQ ID NO.3 encodes.
In one embodiment of the invention, mutant W123K gene as shown in SEQ ID NO.5 encodes.
In one embodiment of the invention, mutant D94A/W123K gene as shown in SEQ ID NO.7
Coding.
Third object of the present invention is to provide the carrier for carrying the gene, including but not limited to plasmid, bacteriophage or
Viral vectors.
In one embodiment of the invention, the carrier is pET serial carrier, such as pET-28a.
Fourth object of the present invention is to provide a kind of raising meso-diaminopimelate dehydrogenase catalytic activity side
Method is that the 94th aspartic acid is replaced with alanine on the basis of SEQ ID NO.2, or/and by the 123rd tryptophan
It is mutated into lysine.
The meso-diaminopimelate dehydrogenase mutant is expressed fifth object of the present invention is to provide a kind of
Cell.
In one embodiment of the invention, the cell is host with bacterium or fungal cell, meets recombination table
Stablize the condition of self-replacation up to carrier, and the meso-diaminopimelate dehydrogenase mutant gene can be made effective
Expression.
In one embodiment of the invention, the host of the cell includes but is not limited to E.coli BL21, E.coli
BL21 (DE3), E.coli JM109, E.coli DH5 α or E.coli TOP10.
In one embodiment of the invention, the genetic engineering bacterium is with Escherichia coli (Escherichia coli)
BL21 (DE3) is host.
Meso-diaminopimelate dehydrogenase method is produced sixth object of the present invention is to provide a kind of, is training
Above-mentioned cell is supported, induction obtains meso-diaminopimelate dehydrogenase mutant protein.
In one embodiment of the invention, the culture is by genetic engineering bacterium in LB culture medium, at 35-39 DEG C
It cultivates to OD600Reach 0.6-0.9, final concentration of 0.1-1.0mM isopropyl-beta D-thio galactopyranoside (IPTG) is added
The induction of solution, in 15-17 DEG C of Fiber differentiation 12-16h.
In one embodiment of the invention, the LB culture medium contains albumen arteries and veins 10g/L, yeast extract 5g/L, chlorination
Sodium 10g/L, pH 7.2.
7th purpose of the invention is to provide above-mentioned meso-diaminopimelate dehydrogenase mutant or above-mentioned cell
Prepare D-phenylalanine or product containing D-phenylalanine in application.
Beneficial effects of the present invention:
It is prominent that the present invention carries out fixed point to amino acid residue in meso-diaminopimelate dehydrogenase substrate channels for the first time
Become, the binding site in the substrate channels of enzyme has been transformed, obtains to have in more high catalytic activity substrate phenylpyruvic acid and disappear
Rotation-diaminopimelate dehydrogenase.The meso that catalytic efficiency provided by the invention improves-diaminopimelate dehydrogenase mutation
Body, wherein simple point mutation D94A, W123K has been respectively increased 3.5 and 1.5 times for the catalytic efficiency of phenylpyruvic acid, complex mutation
Body D94A/W123K improves 1.3 times, and simple point mutation W148K is (on the basis of SEQ ID NO.2, by the 148th tryptophan
It is mutated into lysine, obtains mutant Trp148Lys, i.e. W148K, containing amino acid sequence shown in SEQ ID NO.10, is compiled
Its nucleotide sequence of code is as shown in SEQ ID NO.9) it is only the 80% of wild type for the catalytic efficiency of phenylpyruvic acid.
Specific embodiment
Enzyme-activity unit (U) is defined as: at 50 DEG C, enzyme amount needed for 1 μm of ol NADPH oxidation of catalysis per minute.
Specific enzyme activity is enzyme activity (U/mg) possessed by every milligram of albumen.
Enzyme activity determination reaction system is (100 μ L): 0.2mM NADPH, 2mM phenylpyruvic acid, 200mM glycine-hydroxide
Sodium buffer (pH 9.0), 200mM ammonium chloride buffer (pH 9.0) and suitable pure enzyme.
Embodiment 1: meso-diaminopimelate dehydrogenase mutant building
To contain the meso-from thermophilic ball-type bacillus (Ureibacillus thermosphaericus)
PET-28a recombinant plasmid (the method connected using digestion of diaminopimelate dehydrogenase gene (as shown in SEQ ID NO.1)
Building obtains) as template.
Design upstream and downstream primer using the oligonucleotide fragment containing catastrophe point as homology arm, specific primer it is following (overstriking and
Underscore is mutational site):
Mutant plasmid is constructed using full plasmid inverse PCR method.
PCR amplification system: 1 μ L of template, each 0.5 μ L, 2 × Phanta Max Master Mix polymerase of upstream and downstream primer
10 μ L, sterilize ddH28 μ L of O, 20 μ L of overall reaction system.PCR reaction condition: 95 DEG C of initial denaturations, 5min;95 DEG C of denaturation, 30s, 58
DEG C annealing, 30s, 72 DEG C extension, 2min, 30 circulation;72 DEG C sufficiently extend, 10min.
PCR product is examined by gel electrophoresis, and the Dpn I restriction enzyme of 1 μ L is then added in the PCR product of 15 μ L
Enzyme digests template plasmid, in 25 DEG C stand overnight or 37 DEG C at incubate 3-4h.
The above-mentioned PCR product by digestion process is converted into e. coli bl21 (DE3), obtains recombinating accordingly big
Enterobacteria is coated on the plate containing kanamycins, overnight incubation at 37 DEG C, random picked clones carry out bacterium colony PCR identification and
Sequence verification, the results showed that successfully turn containing meso-diaminopimelate dehydrogenase mutant gene recombinant expression carrier
Change into expressive host e. coli bl21 (DE3).Through sequence verification, glycerol is added and in -40 being mutated successful bacterium solution kind
DEG C Storage in refrigerator.It is final to obtain meso-diaminopimelate dehydrogenase mutant D94A, W123K, D94A/W123K, W148K
Nucleotide sequence sequencing result respectively as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9,
The protein amino acid sequence of corresponding encoded such as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10
It is shown.
Embodiment 2: meso-diaminopimelate dehydrogenase mutant inducing expression
Meso-diaminopimelate dehydrogenase mutant engineering bacteria that embodiment 1 constructs is seeded to containing 50 μ g/mL cards
In the LB liquid medium of that mycin, 37 DEG C, after 180r/min overnight incubation switching in the LB culture medium of 50mL.Inoculum concentration
1%, 37 DEG C of cultivation temperature, revolving speed 180r/min.It cultivates to OD600Reach the IPTG that final concentration of 0.5mM is added after 0.6-0.9
It is induced, inducing temperature is reduced to 16 DEG C, and after inducing 14h, 4 DEG C, 8000rpm is centrifuged 10min and collects thallus, in -70 DEG C of ice
Case storage is spare.
Embodiment 3: meso-diaminopimelate dehydrogenase mutant isolates and purifies
The wet thallus cell that Example 2 is collected washs secondary, resuspension with the 50mM PBS buffer solution of the pH 7.5 of 10mL
In the 50mM PBS buffer solution of the pH 7.5 of 10mL, oscillation shakes up and is crushed under postposition ultrasonic wave, breaks 1s, stops 3s, total duration
15min.Clasmatosis liquid removes cell fragment in 12000rpm centrifugation 20min, collects supernatant, that is, crude enzyme liquid and utilizes 0.22 μ
It is purified after m membrane filtration for the later separation of enzyme.
Purification column is Ni-NTA column, and packed column volume 5mL first uses loading equilibration buffer M0(20mM Tris, 500mM
NaCl, pH 7.4) balance Ni-NTA column, with the rate loading crude enzyme liquid of 0.5mL/min, with loading equilibration buffer M0Elution with
Unadsorbed albumen is removed, elution buffer M is finally used700(20mM Tris, 500mM NaCl and 700mM imidazoles, pH 7.4)
Target protein is collected in elution, and the enzyme solution of gained after purification is spare in -40 DEG C of storages.
Embodiment 4: the specific enzyme activity of the wild enzyme of meso-diaminopimelate dehydrogenase and its mutant
The enzyme solution after purification that embodiment 3 is obtained carries out passing through enzyme using phenylpyruvic acid as the measurement of the specific enzyme activity of substrate
It is living to calculate meso-diaminopimelate dehydrogenase reduction amination that mark instrument detects the variation of NADPH light absorption value at 340nm
Power.The result shows that using phenylpyruvic acid as substrate, the specific enzyme activity of meso-diaminopimelate dehydrogenase wild type (WT) is
0.67U/mg, the specific enzyme activity of mutant D94A, W123K and D94A/W123K, W148K be respectively 2.38U/mg, 0.99U/mg,
0.92U/mg、0.57U/mg。
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>meso-diaminopimelate dehydrogenase mutant that a kind of catalytic efficiency improves
<160> 16
<170> PatentIn version 3.3
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20 25 30
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35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
50 55 60
Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Asp Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Lys Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Trp Gly Lys Gly Val Ser Leu Gly His Ser Gly Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Ile Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Met Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
225 230 235 240
Lys Gln Asn His Ser Gly Met Pro Asn Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
260 265 270
Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
290 295 300
Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
305 310 315 320
Leu Arg Lys Glu Leu Leu
325
<210> 7
<211> 981
<212> DNA
<213>artificial sequence
<400> 7
atgtctaaaa tccgtatcgg tatcgttggt tacggtaacc tgggtcgtgg tgttgaagct 60
gctatccagc agaacccgga catggaactg gttgctgttt tcacccgtcg tgacccgaaa 120
accgttgctg ttaaatctaa cgttaaagtt ctgcacgttg acgacgctca gtcttacaaa 180
gacgaaatcg acgttatgat cctgtgcggt ggttctgcta ccgacctgcc ggaacagggt 240
ccgtacttcg ctcagtactt caacaccatc gactctttcg ccacccacgc tcgtatcccg 300
gactacttcg acgctgttaa cgctgctgct gaacagtctg gtaaagttgc tatcatctct 360
gttggtaagg acccgggtct gttctctctg aaccgtctgc tgggtgaagt tgttctgccg 420
gttggtaaca cctacacctt ctggggtaaa ggtgtttctc tgggtcactc tggtgctatc 480
cgtcgtatcc agggtgttaa aaacgctgtt cagtacatca tcccgatcga cgaagctgtt 540
aaccgtgttc gttctggtga aaacccggaa ctgtctaccc gtgaaaaaca cgctatggaa 600
tgcttcgttg ttctggaaga aggtgctgac ccggctaaag ttgaacacga aatcaaaacc 660
atgccgaact acttcgacga atacgacacc accgttcact tcatctctga agaagaactg 720
aaacagaacc actctggtat gccgaacggt ggtttcgtta tccgttctgg taaatctgac 780
gaaggtcaca aacagatcat cgaattctct ctgaacctgg aatctaaccc gatgttcacc 840
tcttctgctc tggttgctta cgctcgtgct gcttaccgtc tgtctcagaa cggtgacaaa 900
ggtgctaaaa ccgttttcga catcccgttc ggtctgctgt ctccgaaatc tccggaagac 960
ctgcgtaaag aactgctgta a 981
<210> 8
<211> 326
<212> PRT
<213>artificial sequence
<400> 8
Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg
1 5 10 15
Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala
20 25 30
Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val
35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
50 55 60
Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Ala Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Lys Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Trp Gly Lys Gly Val Ser Leu Gly His Ser Gly Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Ile Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Met Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
225 230 235 240
Lys Gln Asn His Ser Gly Met Pro Asn Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
260 265 270
Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
290 295 300
Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
305 310 315 320
Leu Arg Lys Glu Leu Leu
325
<210> 9
<211> 981
<212> DNA
<213>artificial sequence
<400> 9
atgtctaaaa tccgtatcgg tatcgttggt tacggtaacc tgggtcgtgg tgttgaagct 60
gctatccagc agaacccgga catggaactg gttgctgttt tcacccgtcg tgacccgaaa 120
accgttgctg ttaaatctaa cgttaaagtt ctgcacgttg acgacgctca gtcttacaaa 180
gacgaaatcg acgttatgat cctgtgcggt ggttctgcta ccgacctgcc ggaacagggt 240
ccgtacttcg ctcagtactt caacaccatc gactctttcg acacccacgc tcgtatcccg 300
gactacttcg acgctgttaa cgctgctgct gaacagtctg gtaaagttgc tatcatctct 360
gttggttggg acccgggtct gttctctctg aaccgtctgc tgggtgaagt tgttctgccg 420
gttggtaaca cctacacctt caagggtaaa ggtgtttctc tgggtcactc tggtgctatc 480
cgtcgtatcc agggtgttaa aaacgctgtt cagtacatca tcccgatcga cgaagctgtt 540
aaccgtgttc gttctggtga aaacccggaa ctgtctaccc gtgaaaaaca cgctatggaa 600
tgcttcgttg ttctggaaga aggtgctgac ccggctaaag ttgaacacga aatcaaaacc 660
atgccgaact acttcgacga atacgacacc accgttcact tcatctctga agaagaactg 720
aaacagaacc actctggtat gccgaacggt ggtttcgtta tccgttctgg taaatctgac 780
gaaggtcaca aacagatcat cgaattctct ctgaacctgg aatctaaccc gatgttcacc 840
tcttctgctc tggttgctta cgctcgtgct gcttaccgtc tgtctcagaa cggtgacaaa 900
ggtgctaaaa ccgttttcga catcccgttc ggtctgctgt ctccgaaatc tccggaagac 960
ctgcgtaaag aactgctgta a 981
<210> 10
<211> 326
<212> PRT
<213>artificial sequence
<400> 10
Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg
1 5 10 15
Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala
20 25 30
Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val
35 40 45
Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp
50 55 60
Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly
65 70 75 80
Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Asp Thr His
85 90 95
Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln
100 105 110
Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe
115 120 125
Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr
130 135 140
Tyr Thr Phe Lys Gly Lys Gly Val Ser Leu Gly His Ser Gly Ala Ile
145 150 155 160
Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Ile Ile Pro Ile
165 170 175
Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser
180 185 190
Thr Arg Glu Lys His Ala Met Glu Cys Phe Val Val Leu Glu Glu Gly
195 200 205
Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr
210 215 220
Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu
225 230 235 240
Lys Gln Asn His Ser Gly Met Pro Asn Gly Gly Phe Val Ile Arg Ser
245 250 255
Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn
260 265 270
Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala
275 280 285
Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr
290 295 300
Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp
305 310 315 320
Leu Arg Lys Glu Leu Leu
325
<210> 11
<211> 42
<212> DNA
<213>artificial sequence
<400> 11
ctctttcgcc acccacgctc gtatcccgga ctacttcgac gc 42
<210> 12
<211> 44
<212> DNA
<213>artificial sequence
<400> 12
gagcgtgggt ggcgaaagag tcgatggtgt tgaagtactg agcg 44
<210> 13
<211> 42
<212> DNA
<213>artificial sequence
<400> 13
ctgttggtaa ggacccgggt ctgttctctc tgaaccgtct gc 42
<210> 14
<211> 45
<212> DNA
<213>artificial sequence
<400> 14
acccgggtcc ttaccaacag agatgatagc aactttacca gactg 45
<210> 15
<211> 42
<212> DNA
<213>artificial sequence
<400> 15
caccttcaag ggtaaaggtg tttctctggg tcactctggt gc 42
<210> 16
<211> 45
<212> DNA
<213>artificial sequence
<400> 16
cacctttacc cttgaaggtg taggtgttac caaccggcag aacaa 45
45
Claims (10)
1. a kind of meso-diaminopimelate dehydrogenase mutant, which is characterized in that the mutant be (a) or (b) or
(c):
(a) containing amino acid sequence shown in SEQ ID NO.2;
Or, (b) containing amino acid sequence shown in SEQ ID NO.4;
Or, (c) containing amino acid sequence shown in SEQ ID NO.6.
2. encoding meso-diaminopimelate dehydrogenase mutant gene described in claim 1.
3. carrying the carrier of gene described in claim 2.
4. carrier as claimed in claim 3, which is characterized in that the carrier is pET serial carrier.
5. a kind of raising meso-diaminopimelate dehydrogenase catalytic activity method, which is characterized in that be in SEQ ID
On the basis of NO.2, the 94th aspartic acid is replaced with into alanine, or/and the 123rd tryptophan is mutated into lysine.
6. expressing meso-diaminopimelate dehydrogenase mutant cell described in claim 1.
7. cell as claimed in claim 6, which is characterized in that the cell is with Escherichia coli (Escherichia coli)
BL21 (DE3) is host.
8. a kind of produce meso-diaminopimelate dehydrogenase method, which is characterized in that described in culture claim 6 or 7
Cell, induction obtain meso-diaminopimelate dehydrogenase.
9. method according to claim 8, which is characterized in that the culture is placed in cell in LB culture medium, 35-39 DEG C
It is lower to cultivate to OD600Reach 0.6-0.9, final concentration of 0.1-1.0mM isopropyl-beta D-thio galactopyranoside solution be added,
In 15-17 DEG C of Fiber differentiation 12-16h.
10. meso described in claim 1-diaminopimelate dehydrogenase mutant or the cell of claim 6 or 7 are being made
Application for D-phenylalanine or in the product containing D-phenylalanine.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112746061A (en) * | 2021-02-23 | 2021-05-04 | 江南大学 | Meso-diaminopimelate dehydrogenase mutants and uses thereof |
CN112921012A (en) * | 2021-03-18 | 2021-06-08 | 江南大学 | Corynebacterium glutamicum meso-2, 6-diaminopimelate dehydrogenase mutant and application thereof |
CN115786296A (en) * | 2022-09-27 | 2023-03-14 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
Citations (1)
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JP2017108740A (en) * | 2015-12-15 | 2017-06-22 | 国立研究開発法人産業技術総合研究所 | Modified meso-diaminopimelic acid dehydrogenase |
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JP2017108740A (en) * | 2015-12-15 | 2017-06-22 | 国立研究開発法人産業技術総合研究所 | Modified meso-diaminopimelic acid dehydrogenase |
Non-Patent Citations (3)
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HIRONAGA AKITA等: "Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application", 《FRONTIERS IN MICROBIOLOGY》 * |
HIRONAGA AKITA等: "Highly stable meso-diaminopimelate dehydrogenase from an Ureibacillus thermosphaericus strain A1 isolated from a Japanese compost: purification, characterization and sequencing", 《AMB EXPRESS》 * |
JUNJI HAYASHI等: "Structure-Based Engineering of an Artificially Generated NADP+-Dependent D-Amino Acid Dehydrogenase", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112746061A (en) * | 2021-02-23 | 2021-05-04 | 江南大学 | Meso-diaminopimelate dehydrogenase mutants and uses thereof |
CN112921012A (en) * | 2021-03-18 | 2021-06-08 | 江南大学 | Corynebacterium glutamicum meso-2, 6-diaminopimelate dehydrogenase mutant and application thereof |
CN112921012B (en) * | 2021-03-18 | 2022-10-11 | 江南大学 | Corynebacterium glutamicum meso-2, 6-diaminopimelate dehydrogenase mutant and application thereof |
CN115786296A (en) * | 2022-09-27 | 2023-03-14 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
CN115786296B (en) * | 2022-09-27 | 2024-01-30 | 山东理工大学 | Meso-diaminopimelate dehydrogenase mutant and production method thereof |
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